Clathrin-coated vesicles (ccv) play important roles in sorting plasma membrane proteins into the endocytic pathway and sorting proteins between the trans Golgi network (TGN) and endosomes. These ccv-mediated pathways are fundamental, conserved elements of eukaryotic cells; pathway defects can cause inherited human disorders and are likely to contribute to multigenic diseases such as cancer and heart disease. The overall goal of this project is to understand the molecular basis of selective protein transport by ccv in normal cells to provide a foundation for understanding how defects can lead to disease. Towards this goal ccv-mediated protein transport has been characterized in the yeast Saccharomyces cerevisiae. During the previous funding period the actin associated protein Sla1p was identified as a sorting signal recognition factor for NPFX(1,2)D endocytic sorting signals and the Sla1p SHD1 region was defined as a novel sorting signal binding domain. Also, a physiological role for SHD1 in the temporal/spacial regulation of cell wall synthesis was discovered. Based on these advances, genetic, genomic, biochemical, structural, and cell biological approaches will be applied to these specific aims: 1) define the mechanism of cargo recognition by Sla1p during endocytosis; 2) determine the role of Sla1p SHD1 in down-regulating cell wall synthesis; 3) characterize phospho-regulation of SHD1 activity by members of the PKB/Akt kinase subfamily; 4) identify new components of clathrin-mediated endocytic and TGN/endosome trafficking pathways. Together these studies are expected to provide significant advances in understanding the molecular basis and regulation of cargo selection in endocytosis, roles that selective protein recognition can play in cellular physiology, and functions of novel components of the clathrin-based transport machineries that act at the plasma membrane, TGN, and endosomes.